Cytosine DMA methylation is an epigenetic mark for gene silencing that is important in many gene regulatory systems including genomic imprinting, X-chromosome inactivation, and the silencing of transposons and other DNA sequences containing either direct or inverted repeats. Methylation is important in cancer biology, as tumors often show both genome wide demethylation and hypermethylation of specific tumor suppressor genes. We are studying the mechanisms of DNA methylation control in the model plant Arabidopsis thaliana. Arabidopsis DNA methylation systems have much in common with mammalian systems, showing homologs of the three human DNA methyltransferases, Dnmtl, 2, and 3. Both forward and reverse genetics can be performed, and Arabidopsis can tolerate mutations that virtually eliminate methylation, allowing for detailed analysis. We have recently discovered that RNA silencing components including a DICER, an RNA-dependent RNA polymerase, and an ARGONAUTE protein, are central to the mechanisms by which DNA methylation is established, and by which methylation in non-CG contexts is maintained. We propose a series of genetic and biochemical experiments to further study this phenomena. Direct repeats and inverted repeats can trigger de novo DNA methylation by different mechanisms, and we propose to study these differences. We also propose mutant screens aimed at isolating factors important in these processes. The ARGONAUTE4 protein likely acts at the interface between small RNAs and chromatin modifying enzymes, and we propose to study the mechanism of action of this protein in detail. Since many aspects of DNA methylation control mechanisms are similar in diverse eukaryotic organisms, it is likely that progress made in Arabidopsis will shed light on a wide array of epigenetic regulatory systems involved in genome defense, developmental biology, and human disease.